A major contribution to ocular morbidity is lacrimal dysfunction, affecting over 10 million Americans, primarily women. At least two to four million cases are autoimmune-mediated and accompanied by additional symptoms, which lead to the diagnosis of Sjogren's syndrome. Development of primary lacrimal deficiency and Sjogren's syndrome are associated with changes in hormonal status and also environmental and genetic factors. Although the precise mechanisms involved in disease development remain unclear, these diseases are associated with impaired release of secretory products into ocular surface fluid by the lacrimal gland. The principal cell of the lacrimal gland and primary contributor of proteins into ocular fluid is the lacrimal acinar cell. Disease-related changes in release of secretory products into ocular fluid imply, at some level, changes in the molecular mechanisms responsible for exocytosis at the apical plasma membrane of lacrimal acini. Our approach to the study of diseases affecting lacrimal gland secretory functions has therefore focused on elucidating the mechanisms responsible for accurate exocytotic release of secretory pro- ducts at the apical plasma membrane of healthy lacrimal acini, and to determine how changes in these mechanisms are related to the establishment and progression of lacrimal disease. Findings during the previous project period have defined the involvement of several key effectors of exocytosis including rab3D, vesicle-associated membrane protein 2, cytoplasmic dynein and actin filaments. Moreover, compensatory retrieval of acinar apical plasma membrane, essential for regeneration of secretory vesicles, utilizes a unique clathrin-mediated endocytotic mechanism involving actin, syndapins and N-WASP. Key questions regarding other fundamental mechanisms of lacrimal acinar exocytotic pathways remain unanswered, and will be addressed in Aims #1 and #2 using reconstituted primary rabbit lacrimal acini as an experimental model. To begin to address the changes in exocytotic pathways associated with initiation and progression of Sjogren's syndrome, Aim #3 probes age- and sex-related changes in organization and composition of the exocytotic pathway in lacrimal glands from control BALB/c mice as well as NOD mice, an experimental model of Sjogren's syndrome. Technically, these aims utilize an array of techniques including light and electron microscopy, biochemical analysis of subcellular membranes and proteomics. The specific aims are: Aim #1. Do lacrimal acinar cells contain distinct secretory vesicle populations? Aim #2. How do actin filaments facilitate exocytosis of secretory vesicles in lacrimal acinar cells? Aim #3. What components of the exocytotic pathway undergo age-related changes in lacrimal glands from BALB/c and NOD mice?